Conditions will be established for long-term (30-day minimum) culturing of mass numbers of skeletal muscle fibers derived from primary cultures of chick embryo cells. These cultures will be cocultured with motor neurons differentiating from neuronal cell lines. Neuronal cell lines will be maintained as stocks. We will attempt to establish within long-term cultures attachment sites for muscle fibers which more faithfully mimic normal myotendenous junctions. Electrical stimulation of cultures, with and without nerve cells will be carried out. We will define biochemical responses of muscle fibers to (a) longterm culture in the absence of innervaton, (b) innervation by neuronal cell lines, (c) electrical stimulation at fast and slow muscle motor neuron frequencies, in the absence of innervaiton, (d) electrical stimulation in the presence of innervation. The major biochemical changes that will be monitored will be (1) the synthesis and turnover rates of myosin heavy chain by radioimmune assay combined with 1-D, SDS electrophoresis, (2) synthesis and accumulation of other myofibrillar peptides by pulse-chase combined with 2-D gel electrophoresis and specific antibody staining, (3) the synthesis and accumulation of mRNA's coding for a variety of myofibrillar peptides by translation in reticulocyte lysates combined with radioimmune assay and by quantitative hybridization (when possible) of muscle culture mRNA to cDNA probes. Particular attention will be paid to whether or not innervation, electrical stimulation, or aging produce changes in isoforms of myosin, especially light chains and alternative forms of tropomyosins.